A Unique β-1,2-Mannosyltransferase of Thermotoga maritima That Uses Di- myo -Inositol Phosphate as the Mannosyl Acceptor

Abstract

In addition to di-myo-inositol-1,3-phosphate (DIP), a compatible solute widespread in hyperthermophiles, the organic solute pool of Thermotoga maritima comprises 2-(O-␤-D-mannosyl)-di-myo-inositol-1,3-phosphate (MDIP) and 2-(O-␤-D-mannosyl-1,2-O-␤-D-mannosyl)-di-myo-inositol-1,3-phosphate (MMDIP), two newly identified ␤-1,2-mannosides. In cells grown under heat stress, MDIP was the major solute, accounting for 43% of the total pool; MMDIP and DIP accumulated to similar levels, each corresponding to 11.5% of the total pool. The synthesis of MDIP involved the transfer of the mannosyl group from GDP-mannose to DIP in a single-step reaction catalyzed by MDIP synthase. This enzyme used MDIP as an acceptor of a second mannose residue, yielding the di-mannosylated compound. Minor amounts of the tri-mannosylated form were also detected. With a genomic approach, putative genes for MDIP synthase were identified in the genome of T. maritima, and the assignment was confirmed by functional expression in Escherichia coli. Genes with significant sequence identity were found only in the genomes of Thermotoga spp., Aquifex aeolicus, and Archaeoglobus profundus. MDIP synthase of T. maritima had maximal activity at 95°C and apparent K m values of 16 mM and 0.7 mM for DIP and GDP-mannose, respectively. The stereochemistry of MDIP was characterized by isotopic labeling and nuclear magnetic resonance (NMR): DIP selectively labeled with carbon 13 at position C1 of the L-inositol moiety was synthesized and used as a substrate for MDIP synthase. This ␤-1,2-mannosyltransferase is unrelated to known glycosyltransferases, and within the domain Bacteria, it is restricted to members of the two deepest lineages, i.e., the Thermotogales and the Aquificales. To our knowledge, this is the first ␤-1,2-mannosyltransferase characterized thus far.Thermotoga maritima was first isolated from hot marine sediments on Vulcano Island, Italy, being able to grow between 55°C and 90°C (14). This strictly anaerobic bacterium ferments a variety of simple and complex carbohydrates to acetate, hydrogen, and CO 2 (10). In line with these metabolic traits, a substantial percentage of the genes annotated in the genome of this hyperthermophile are allocated to the metabolism of mono-and polysaccharides (8, 23). Therefore, T. maritima has been pointed out as a source of glycoside hydrolases with potential industrial relevance, namely, in processes of conversion of biomass into biofuels (3, 34).Like many other hyperthermophiles isolated from marine environments, Thermotoga maritima is slightly halophilic (optimum NaCl concentration of 2.7%, wt/vol) and has developed biochemical strategies to counterbalance the external osmotic pressure. The accumulation of low-molecular-mass organic compounds in the cytoplasm is the most common osmoadaptation mechanism, which enables a rapid response to fluctuations in the salinity of the external medium. Interestingly, the organic solutes encountered in organisms adapted to thrive in hot environments are clearly different from those ...

Thermococcus kodakar ensis Mutants Deficient in Di- myo -Inositol Phosphate Use Aspartate To Cope with Heat Stress

“…Moreover, there are several examples of solutes from hyperthermophiles whose levels respond both to heat and salt stress (31). For instance, ␣-glutamate accumulates in Thermotoga maritima in response to these two types of stress (27).…”

“…The data reveal a trend toward specialization of roles in thermoadaptation and osmoadaptation. Indeed, mannosylglycerate and diglycerol phosphate typically accumulate in response to increased NaCl concentration in the growth medium, whereas the levels of DIP and derivatives consistently increase at supraoptimal growth temperatures (11,16,17,27,31). …”

“…Mannosylglycerate, mannosylglyceramide, di-myo-inositol phosphate, mannosyldi-myo-inositol phosphate (DIP), diglycerol phosphate, and glycero-phospho-myo-inositol are examples of compatible solutes highly restricted to thermophiles and hyperthermophiles (27,31). Our team has, over several years, examined the compatible solute composition in a large number of hyperthermophiles and their accumulation under stressful conditions.…”

Insights into thermoadaptation and the evolution of mesophily from the bacterial phylum Thermotogae

“…Moreover, in the bacterium Calderobacterium hydrogenophilum polyamine compounds stabilize the 70S initiation complex of ribosomes (Mikulik and Anderova 1994). Many temperature studies in the Thermotogae have focused on the accumulation of these organic compounds and polyamines and the elucidation of their biosynthetic pathways in T. maritima and the more moderate thermophile Petrotoga miotherma (Zellner and Kneifel 1993;Jorge et al 2007;Rodrigues et al 2009;Oshima et al 2011;Rodionova et al 2013). Several compatible solutes have so far been found only in thermophiles, including di-myoinositol phosphate, mannosyl-di-myo-inositol phosphate, mannosylglyceramide, and diglycerol phosphate (Borges et al 2010;Gonçalves et al 2012), and novel thermophilic solutes continue to be identified (Jorge et al 2007;Rodrigues et al 2009).…”

“…When the ability to synthesize di-myo-inositol phosphate was removed from Thermococcus kodakarensis by deleting a key synthesis gene, the growth of this archaeon was unaffected, and aspartate accumulated as an alternative compatible solute (Borges et al 2010). In the Thermotogae, multiple solutes accumulate under stress conditions (Jorge et al 2007;Rodrigues et al 2009). This suggests that although the role compatible solutes play in thermal protection is not fully understood, there is functional redundancy among the solutes.…”